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Abstract

Background

Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic
composition in the chemical and materials sciences, but its use in biology has been
limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS),
which combines a new generation of SIMS instrument with sophisticated ion optics,
labeling with stable isotopes, and quantitative image-analysis software, was developed
to study biological materials.

Results

The new instrument allows the production of mass images of high lateral resolution
(down to 33 nm), as well as the counting or imaging of several isotopes simultaneously.
As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus
of the levels of enrichment in specific isotopic labels, within volumes of less than
a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few
atomic layers are needed to create an atomic mass image. We illustrate the use of
MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid
transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic
kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus
using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes
12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse.

Conclusion

MIMS makes it possible for the first time to both image and quantify molecules labeled
with stable or radioactive isotopes within subcellular compartments.

Background

The fundamental discovery that proteins in biological tissues are in a dynamic state
was made in the late 1930s using a custom-built mass spectrometer to measure the incorporation
into proteins of the stable nitrogen isotope 15N [1], which was provided in the mouse diet as 15N-leucine and used as a marker of amino acids. These seminal studies could not be
pursued at the subcellular level because there was no methodology to simultaneously
image and quantitate a stable isotope and because there is no meaningful radioactive
isotope of nitrogen. Imaging of stable-isotope distribution has been possible, however,
since the development of mass filtered emission ion microscopy using secondary ions
by Castaing and Slodzian [2], which is part of the technique later named secondary-ion mass spectrometry (SIMS).
With this technique, a beam of ions (the primary-ion beam) is used as a probe to sputter
the surface atomic layers of the sample into atoms or atomic clusters, a small fraction
of which are ionized (Figure 1) [3]. These secondary ions, which are characteristic of the composition of the region
analyzed, can be manipulated with ion optics just as visible light can be with glass
lenses and prisms. In a SIMS instrument, the secondary ions are separated according
to mass and then used to measure a secondary-ion current or to create a quantitative
atomic mass image of the analyzed surface. SIMS has become a major tool in semiconductor
and surface-science studies [4], geochemistry [5,6], the characterization of organic material [7], and cosmochemistry [8,9].

Figure 1. The principle of secondary-ion mass spectrometry. The primary Cs+ beam hits the sample and sputters the surface. Atoms and molecular fragments are ejected
from the sample surface; during this process a fraction of the secondary particles
are ionized. The identity of the secondary particles, determined by mass spectrometry,
indicates the atoms or atomic clusters from the molecules in the sample that have
been hit by the primary Cs+ beam. The figure shows only the types of atoms and ions that are relevant to this
article; other particles formed by sputtering are not represented. Cs, cesium.

Although there has been pioneering work using SIMS in biology [10-14], SIMS technology, until now, has presented irreconcilable tradeoffs [15] that have severely limited its use as a major discovery tool in biomedical research.
To make secondary-ion methodology practicable for locating and measuring isotope tags
in subcellular volumes, four major issues need to be addressed. First, to produce
quantitative ultrastructural images, the technique must have sufficiently high spatial
resolution, and quantitation and imaging must be associated. Second, because the quantitation
of label involves measuring the excess of an isotope tag above its natural occurrence,
and this excess is calculated by the ratio of two isotopes, the data from the two
isotopes should be recorded simultaneously, in parallel, and from exactly the same
region of the sample (that is, in register). This is to ensure that changes in instrument
or sample conditions do not lead to errors in the calculated isotope ratios. Third,
because nitrogen has an electron affinity of zero, N- ions do not form; nitrogen must therefore be detected as cyanide ions (CN-). Consequently, in order to use the stable isotope 15N as a label, the mass resolution of the instrument needs to be high enough to separate
the ions 12C15N- and 13C14N-, which have the same mass number of 27, but do not have exactly the same atomic mass
weight: differing by 0.00632 mass unit, less than 1 part in 4,000. Finally, the high
mass resolution should not be at the expense of the secondary-ion current; the transmission
of secondary ions from sample to detector needs to be high enough to allow data collection
from sub-cubic-micrometer volumes and in a reasonable amount of time.

These four requirements necessitate a previously unattainable combination of instrumental
capabilities, with the ability to collect large numbers of secondary ions at high
mass resolution, parallel detection of several secondary ions, high lateral resolution,
and high precision of measurements. A new generation of secondary-ion mass spectrometer
has now been developed that can measure several ion masses in parallel, has a high
mass resolution (mass/change in mass ratio of approximately 10,000) at high secondary-ion
relative transmission (70–80%), and has a high lateral resolution, down to 33 nm [16,17]. In this paper we present some biological applications of this new technology. These
extraordinary capabilities allow us, for example, to image and measure in parallel
the intracellular distribution of molecules labeled with the stable isotopes 15N or 13C because we can separate the isobaric species (that is, the species with the same
atomic masses): 12C15N- from 13C14N- or 13C- from 12C1H-. Because the images are produced in parallel from the same sputtered volume they
are in exact register with each other and these characteristics are necessary for
obtaining quantitative atomic mass images. A quantitative mass image contains at each
pixel a number of counts, which are a measure of the selected atomic mass and are
directly proportional to the selected atomic mass abundance in the sample, at a location
corresponding to the pixel address. Counts from several atomic masses, originating
from the same location in the sample, can be recorded in parallel at the same pixel
address, which allows us to derive meaningful isotope ratios. Isotope ratios are at
the core of the methodology. When the sample has been labeled with a given isotope,
a ratio higher than its natural abundance indicates the presence of the marker isotope
at a particular location as well as measuring its relative excess. In addition, the
high stabilities of the primary beam, the ion optics, the mass spectrometer and the
detectors contribute to very precise measurements.

We have developed the use of this new generation of SIMS, together with tracer methods
and quantitative image-analysis software, for locating and measuring molecules labeled
with stable isotopes in subcellular compartments, a development that we call multi-isotope
imaging mass spectrometry (MIMS). In this paper we present a range of examples showing
how MIMS can be used to provide atomic mass images of biological specimens, and how
in combination with stable isotope labeling it provides qualitative and quantitative
information that is not possible to obtain with other methods.

Results

Imaging of unlabeled cells and tissue sections

One qualitative application of MIMS is high-resolution imaging. Detailed anatomical
images can be obtained from unstained, unlabeled samples using the 12C14N- secondary ion, as shown by the analysis of a section through mouse cochlea (Figure
2a–c). A fixed, unstained section mounted on silicon was first examined using reflection
differential interference contrast (RDIC) microscopy (Figure 2a) to select regions of interest that can be retrieved after the sample is hidden inside
the SIMS instrument. The mass image of the 12C14N- ions sputtered from a 80-μm field corresponding to the boxed area in Figure 2a is shown in Figure 2b, and the mass image of the 12C14N- ions sputtered from a 20-μm subfield of Figure 2b is shown in Figure 2c. The contrast of these mass images provides a very detailed view of the cochlear
structures. Only a few atomic layers of the surface of the sample are sputtered using
the standard analytical conditions (see Discussion and [Additional data file 1]).
Thus, although the method is nominally destructive, we can analyze the same field
repetitively, up to a total of tens of hours or hundreds of scans, without observing
gross morphological alterations (data not shown). A large area can be imaged relatively
quickly in order to select regions of interest for quantitative analysis, as illustrated
by the reconstruction of a mouse cochlea shown in Figure 2d. The mass image of 12C14N- ions is made up of ten tiles, acquired over a total of 20 minutes, with each tile
acquired over an area of 100 × 100 μm in 2 minutes. All the cochlear structures one
would expect to see [18] are visible, and are easy to identify by comparing them with conventional histological
sections.

The high spatial resolution of MIMS is illustrated by its ability to image individual
stereocilia, the mechanosensory organelles of the inner hair cells of the cochlea
(Figure 2e; the field analyzed was 6 × 6 μm). The various intracellular structures are easy
to identify by comparing the image with electron micrographs of the same structure.
We estimate that a lateral resolution better than 33 nm can be achieved using a method
derived from the 'knife-edge' technique (see [Additional data file 2]). Because the
atomic mass image is formed using only a few atomic layers of the sample, the depth
resolution (z resolution) can be smaller than 1 nm, much better than the resolution that would be
provided by an exceptionally thin electron-microscopy section (10 nm). (In this paper,
we define depth resolution as the minimum amount of material that needs to be sputtered
to obtain an atomic mass image.) Studies of the same sections with both MIMS and electron
microscopy – a technique developed for studying cosmic dust [19] – may help provide complementary structural observations.

We do not know from first principles the mechanism of contrast formation in atomic
mass images from unstained samples. A striking observation of intense contrast observed
with MIMS at mass 12C14N, shown in Figure 2g–i, may guide inquiries in this field. We were analyzing a mouse kidney when we observed
a brightly contrasted structure, a circular 'snake', along the lining of the lumen
of an artery. It is likely that this region is the 'elastica interna' as described
in histology texts, a tissue layer that is usually visible only after the arterial
tissue has been specially stained (Figure 2f). The MIMS images of the artery in Figure 2g–i were obtained without this special staining. The images indicate that this structure
produces a high yield of 12C14N- ions in comparison with the other regions of the artery, and suggests a relationship
between the image obtained and molecular composition and density.

MIMS can be used to visualize whole cells as well as sections. These images have a
three-dimensional appearance, showing that MIMS can provide scanning atomic or molecular
ion mass images of samples with relief, as does scanning electron microscopy. The
lamellipodium of a well-spread endothelial cell imaged by MIMS at mass 12C14N is shown in Figure 2j; it appears as a light, sheet-like structure with darker lines radiating from the
cytoplasm to the external border of the lamellipodium. In contrast, if actin polymerization
is blocked by cytochalasin D treatment, the 12C14N mass image of an endothelial cell shows no lamellipodia but only thin, spike-like
projections around the cell circumference, which are most probably retraction fibers
(Figure 2k).

In conclusion, MIMS atomic mass images of CN- ions in biological samples are highly contrasted, even though they are obtained without
any staining. The various structures are easy to identify down to a lateral resolution
of approximately 33 nm, and the depth resolution can be as small as a few atomic layers.
The largest single field that can be imaged is approximately 140 × 140 μm, but larger
fields can be documented quickly by taking a series of images for 1–2 minutes each.
There is no machine-specific requirement for the sample except that a vacuum must
be sustained. Because MIMS is a surface-analysis method, one can use many kinds of
samples: for example, tissue or cell sections embedded in a medium such as epon, or
cells cultured directly on a support that can be brought into the analysis chamber
and prepared with any usual histological technique. The thickness of the sample is
not critical, provided that the electrical charges deposited can be dissipated. The
surface analyzed does not have to be flat, and one can obtain SIMS images of three-dimensional
samples.

Quantitative labeling with stable isotopes

Having established that MIMS can be used to obtain atomic mass images of unstained
biological objects, this led us to develop the unique feature of MIMS: the quantitative
analysis of isotopes within subcellular compartments. We will now discuss how the
technique can be used to measure the incorporation of isotopic tracers within compartments
of sub-cubic-micrometer volume. To do this, the sample is labeled with stable isotopes
such as 15N or 13C, which are present at much lower levels naturally than their counterpart 14N and 12C isotopes, and each isotope is then measured to determine whether it is present in
an amount exceeding its natural abundance. Stable isotope labeling can be used, for
example, to pursue the classic studies of Schoenheimer at the subcellular level [1].

The isotope abundance is measured by recording the secondary-ion currents (counts/time)
obtained from a pair of isotopes, for example, 13C and 12C, calculating the ratio and then comparing it with their natural abundance ratio.
In a control sample, which has not received an excess of the tracer isotope, the counts
of each isotope are related to each other by their natural abundance. In other words,
there will be a count of 13C or of 15N such that 13C/12C = 1.12%, or 15N/14N (measured as 12C15N/12C14N) = 0.367%, calculated from the values of their respective natural abundance. This
means that when measured in parallel, all the analytical conditions being the same,
the 15N (12C15N) count rate will be 272 times lower than the 14N (12C14N) count rate.

Quantitative labeling with 15N

The goal of our first experiments was to ensure that we could measure 15N/14N ratios equivalent to their natural abundance from tiny volumes of untreated control
sample. Our first analyses were carried out using a stationary cesium (Cs+) primary ion beam. We counted in parallel the secondary ions 12C14N- and 12C15N- emitted from various areas smaller than 1 square micrometer in control samples of
mouse tissues. We measured 12C15N/12C14N isotope ratios in control mouse tissue of 0.366% (standard error (SE) = 0.002, n = 12) in the cochlea, 0.368% (SE = 0.001, n = 14) in the kidney, and 0.368% (SE = 0.001, n = 6) in the intestine. These values are not statistically significantly different
from the natural terrestrial value of the 15N/14N isotope ratio, 0.3673% [20]. This proved the feasibility of using this method on biological samples.

We then showed that we could measure the incorporation of a stable isotope label in
an ultra-minute volume of biological material, as done for bulk tissue 60 years ago
[1]. We fed mice a diet slightly enriched with 15N-L-leucine for a sufficient length of time (14 days) to result in total protein renewal
in kidney and intestine. The 15N/14N isotope ratios determined using a stationary primary ion beam at various areas over
the samples were equivalent to the 15N/14N ratio in the diet determined independently by combustion mass spectrometry analysis
(intestine 4.45‰, SE = 0.05, n = 7; kidney 4.41‰, SE = 0.03, n = 12; diet 4.45‰, SE = 0.02, n = 7).

Using this method, only one location can be analyzed at a time and its precise position
is difficult to ascertain in the absence of an image. With our instrument, we have
developed a much more powerful but more complex method of isotope ratio imaging, where
the isotope ratios are calculated from quantitative mass images obtained simultaneously
from a set of isotopes. A quantitative mass image, as we call it, is the representation
of an analyzed field in which each pixel is the address of a register at which the
secondary-ion current of an isotope of interest has been recorded during analysis.
Up to four secondary-ion currents, representative of four isotopes, can be recorded
simultaneously at each pixel address with our instrument, for example 12C-, 13C-, 12C14N- and 12C15N-. A quantitative image of 256 × 256 pixels thus represents a set of (256 × 256 × 4)
or 262,144 numbers. We call a group of pixel addresses a 'region of interest', and
the first step in quantitation is to extract the values of counts/time/isotope from
groups of pixels or from individual pixels. This allows us to measure many more regions
from a single analytical field than we could do using a stationary beam, and also
to associate quantitation and localization among cells and subcellular domains. All
the mass imaging in the rest of this paper will refer to quantitative mass imaging.

We illustrate quantitative mass imaging of 15N with a study of 15N-leucine incorporation in the mouse cochlea, a highly organized tissue with several
different cell types, and in a subcellular structure of this tissue, the stereocilium,
the mechanosensing organelle of hair cells. The secondary-ion mass images of a field
of cochlear tissue from a mouse that has been on a 15N-L-leucine diet for 9 days are shown in Figure 3a–f. Additional data file 3 describes how the quantitative data are extracted from these
images. Mass images of 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. The mass image of the 12C14N- ion (Figure 3a) shows a strikingly detailed histology. 12C14N- ions arise from nitrogen-containing molecules, the most abundant by far being proteins,
which make up 18% of the total weight in most cell types, whereas RNA and DNA make
up 1.1% and 0.25%, respectively [21]. The mass image of the 12C15N- ions (Figure 3b) is similar in form to the 12C14N- image (Figure 3a) but has much lower counts; the total number of counts of 12C15N- ions and of 12C14N- ions are 2.02 × 105 and 4.52 × 107, respectively (note that the subjective brightness of the images is not directly
related to the count rate; see Additional data file 4). The pixel count of the 12C15N- image is a measure of both natural 15N and the supplementary 15N arising from the metabolism of 15N-L-leucine in the cochlea. This supplementary 15N may vary from a minimum of zero to a maximum value equivalent to the 15N added to the diet. The image of the internal control 12C- (Figure 3d) has a relatively poor contrast compared with the 12C14N- image (Figure 3a) because a larger fraction of the 12C- ions arise from the embedding medium, which has a high and uniform carbon content.
The image of the 13C- ions (Figure 3e) is similar to the 12C- image, but with a much lower count rate; the total number of counts of 13C- and of 12C- are 2.56 × 105 and 2.33 × 107, respectively. The pixel counts of the sample resulting in the 13C- ion image contain a mean of 1.10% of the 12C counts, corresponding to the natural ratio of 13C/12C.

Figure 3. MIMS analysis of stereocilia from mice fed 15N-L-leucine. (a-f) Quantitative MIMS images of cochlear hair cells from mice after 9 days on the 15N-L-leucine diet. DC, Deiter cells; OP, outer pillar cells; RL, reticular lamina;
TBC, tympanic border cells (below the basilar membrane); Sb1 and Sb2, stereocilia
bundles; other abbreviations are as in Figure 2. All images are 60 × 60 μm (256 ×
256 pixels) and have an acquisition time of 10 msec/pixel. (a) 12C14N-, (b) 12C15N-, (c) 12C15N-/12C14N- ratio image, (d) 12C-, (e) 13C-, (f) 13C-/12C- ratio image. The images in (c,f) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. Scale bar = 10 μm. (g-l) High-resolution quantitative MIMS images of the stereocilia labeled Sb1 in (a). The
isotopes and ratios shown in each image are indicated and are the same as the equivalent
images in (a-f). All images are 3 × 3 μm (256 × 256 pixels) and an acquisition time
of 40 msec/pixel. Scale bar = 0.5 μm. (m) HSI image of the 12C15N/12C14N ratio derived from (h) and (g). The colors correspond to the excess 15N derived from the measured 12C15N-/12C14N- isotope ratios, expressed as a percentage of the 15N excess in the feed, which is a measure of protein renewal; values range from 0%
(blue) to 60% and higher (magenta). Small magenta areas (α, β, γ, δ, ε, and ζ) indicate
excess 15N. The image is 3 × 3 μm (256 × 256 pixels) and dwell time was 40 msec/pixel. (n) Bar graph of the mean percentage at the stereocilia level of the 15N excess in the feed, which is a measure of protein renewal, after 9 days or 22 days
of 15N-L-leucine diet. L, inter-stereocilia structures; S, core stereocilia at 100–200
nm from L. (o) Bar graph of the mean value of the 13C/12C ratio measured after 9 days at the same locations as in (n). t, value of the natural
terrestrial 13C/12C ratio.

The ratio images 12C15N-/12C14N- (Figure 3c) and 13C-/12C- (Figure 3f) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. The contrast observed in the 12C15N-/12C14N- image is due to the excess of 15N in the area of the cochlea that has incorporated 15N derived from the 15 N-L-leucine. The internal control 13C-/12C- ratio image has no contrast because, in the absence of added 13C, the value of the ratio is equivalent to the natural ratio in any part of the analyzed
field.

Using the quantitative images and the derived ratio images, and guided by the hue
saturation intensity (HSI) transformation (see Materials and methods), we can now
calculate a value for 15N incorporation into the main structures shown in Figure 3a–f. This can be expressed as percentage renewal by comparing the excess 15N in the tissue with the excess 15N in the diet (see Materials and methods). These values represent overall protein
renewal among the different cochlear structures, as demonstrated for whole tissue
in the classic work of Schoenheimer [1]. After 9 days of a 15N-L-leucine diet, the incorporation of 15N is markedly different among specific cell types. The outer hair cells have a 15N renewal of 52.5% ± 1.8 SD, not significantly different from that of the Deiter cells
(47.2% ± 4.8 SD), or of the proximal part of the outer pillar cell above the basilar
membrane (46.0% ± 5.7 SD), and of one population of tympanic border cells (48.3% ±
1.5 SD). The basilar membrane has a small 15N renewal (overall 21.1% ± 6.0 SD), not statistically different from part of the outer
pillar cells at the level of the Deiter cells (18.4% ± 3.7 SD). Overall, the reticular
lamina has a 15N renewal of 30.8% ± 8.9 SD, significantly higher than the basilar membrane and the
distal outer pillar cells, and significantly lower than that of the outer hair cells.
The lone outer hair-cell nucleus observed has a 15N renewal of 35.5%. Finally, we measured a second population of tympanic border cells
with a 15N renewal significantly greater than in any other area (72.8% ± 2.5 SD). The internal
control provided by the epon embedding medium had a 12C15N/12C14N isotope ratio of 0.365% ± 0.089 SD, equivalent to the natural abundance ratio and
corresponding to a 15N renewal of 0%.

The unique power of MIMS is demonstrated by the quantitative imaging of subcellular
structures at high resolution, revealing sub-cubic-micrometer-sized zones with high
15N renewal, and thus probably high protein renewal. In addition, the experiment showed
that the same sample can be analyzed repetitively at a variety of spatial resolutions.
We analyzed one of the bundles of stereocilia (Sb1, indicated by a white arrow in
Figure 3a) at high resolution; we used a field of 3 × 3 μm, a beam size of about 35 nm, and
256 × 256 pixels (Figure 3g–l). Mass images of the 12C-, 13C-, 12 C14N- and 12C15N- ions were acquired in parallel. The 12C14N- image (Figure 3g) shows one bundle of stereocilia and a fraction of the cuticular plate of one hair
cell, barely visible in the lower-resolution image in Figure 3a.

As in the cochlear analysis, but at a subcellular level, the 12C15N- image (Figure 3h) is similar in form to the 12C14N- image (Figure 3g) but has much lower counts; the total number of counts of 12C15N- and of 12C14N- are 8.36 × 104 and 1.61 × 107, respectively. The 12C- image (Figure 3j) has relatively poor contrast compared with the 12C14N- image (Figure 3g), as most of the 12C- ions arise from the embedding medium. The 13C- image (Figure 3k) is similar to the 12C- image, but with a much lower count; the total number of counts for 13C- and 12C- are 4.80 × 105 and 4.36 × 107, respectively. The pixel counts from the 13C- image include the fraction of 13C related to the 12C content by the natural ratio of 13C/12C. The ratio images 12C15N-/12C14N- (Figure 3i) and 13C-/12C- (Figure 3l) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. The contrast observed in the 12C15N-/12C14N- image is due to the excess of 15N in the stereocilia, cuticular plate, and hair-cell areas that have incorporated
15N derived from the 15N-L-leucine. The internal control 13C-/12C- ratio image (Figure 3l) has no contrast, as in Figure 3f.

An HSI transformation of the 12C15N-/12C14N- ratio image of the stereocilia bundle in Figure 3i is shown in Figure 3m. The colors indicate the fractional excess 15N derived from the measured 12C15N-/12C14N- isotope ratios. The HSI image reveals small areas of high excess 15N located towards the tips of stereocilia or between stereocilia (magenta); within
the stereocilia, close to these areas, there is minimal or no excess 15N, as indicated by the predominantly blue-green to blue color.

Guided by the HSI image, we have calculated the values of the 12C15N-/12C14N- ratios and of the percentage 15N renewal for the areas indicated α to ζ and at 100 to 200 nm away from them within
the stereocilia core over an approximately equivalent area (Figure 3m and Table 1). We measured high 15N renewal in areas α to ζ (79.4% ± 12.7 SE, n = 5), whereas at 200 nm away the 15N renewal in stereocilia was very low (4.6% ± 1.27 SE, n = 5). Finally, MIMS allowed us to estimate from the relative counting of mass 12C14N in areas α to ζ and in stereocilia that the above values may have been produced
by objects about 5 nm wide (see also Figure 5k below in the section entitled 'Quantitative labeling of prokaryotic with gaseous
15N). The overall mean values of 15N renewal in structures between stereocilia, found with HSI, and in adjacent stereocilium
cores are shown in Figure 3n. After 9 days on the 15N-L-leucine diet, the mean 15N incorporation into the inter-stereocilia structures was 78.6% ± 10.1 SE (n = 7). In the adjacent stereocilium core (200 nm away), the 15N incorporation was 7.1% ± 2.1 SE (n = 7). After 22 days on the 15N-L-leucine diet, the incorporation of 15N into the inter-stereocilia structures was 100% of its content in the diet, and in
the adjacent stereocilium cores, 15N incorporation was 20.9% ± 3.8 SE (n = 4). In the areas in which 15N values were very different, the internal control 13C/12C ratios (Figure 3o) were very similar between inter-stereocilia structures (1.09% ± 0.04 SE, n = 7) and adjacent stereocilium cores (1.12% ± 0.03 SE, n = 7), and are statistically equivalent to the natural terrestrial ratio of 1.12% [20].

Figure 5. Use of MIMS to study nitrogen-fixing bacteria. (a-c) Secondary ion images from the molecular ions (a) 12C14N-, (b) 12C15N-, and (c) the HSI 12C15N-/12C14N- ratio of a sample containing both Teredinibacter turnerae (Tt; rod-like cells) and Enterococcus faecalis (Ef; bunches of rounded cells) cultured in a 15N atmosphere for 120 h. Field: 46 × 46 μm (512 × 512 pixels); acquisition time 3 min.
The magenta color of the T. turnerae cells is an indication of their incorporation and fixation of 15N (see Figure 3 for explanation). (d) The effect of scaling of the HSI 12C15N-/12C14N- ratio image (the numerator has been multiplied by 100) from T. turnerae cells exposed to a 15N atmosphere for 32 h. Assigning the hue spectrum to the whole range of ratio values
allows easy identification of bacteria most highly enriched in 15N (the turquoise cells in the top left panel). Compressing the hue scale (shown gradually
from top left to lower right) causes images of some of the cells to saturate at the
magenta level and allows us to easily recognize a succession of cells also enriched
in 15N, although at a lower level. The isotope values start with 0–7 (top left; a value
of 7 is 19-fold higher than the natural ratio) and go to 0–0.5 (bottom right; a value
of 0.5 is 1.43 times the natural ratio). The field of view is 13 × 13 μm (256 × 256
pixels); acquisition time 20 min. (e,f) HSI image of the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) of a T. turnerae cell exposed to a 15N atmosphere for 96 h. Field: (e) 8 × 8 μm; (f) 6 × 6 μm. Acquisition time: (e) 10
min; (f) 40 min. (g,h) HSI image of (g) the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) and (h) the 13C-/12C- ratio of T. turnerae in shipworm gill bacteriocytes incubated in the presence of a 15N atmosphere for 4 h. Field: 10 μm × 10 μm (256 × 256 pixels); acquisition time 60
min. (i,j) HSI image (i) of the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) and (j) at 12C15N- of T. turnerae exposed for 96 h in a 15N atmosphere. Arrows indicate the flagella of the bacteria. Field: 60 × 60 μm (256
× 256 pixels); acquisition time 20 min. (k) Line scan across the flagellum observed in (i,j) showing 12C15N- secondary-ion counts as a function of pixel address across the flagellum. One pixel
is equivalent to 234 nm. Inset: arrow points to the flagellum; the red box indicates
the area of the bacterium that was used to evaluate the mean 12C15N- counts.

We can thus measure with high precision in a single analyzed field a variety of values
of 15N incorporation among different cell types, as calculated from the quantitative mass
images in Figure 3a–c, or among subcellular structure over an area of 9 μm2 square, as calculated from the quantitative mass images in Figure 3g–i.

Quantitative labeling with 13C

Despite the importance of free fatty acids (FFAs) for life, studies of their transport
are difficult to extend to the cellular scale because no suitable methodology is available.
Autoradiography cannot provide quantitative information on accumulation of FFAs in
intracellular fat droplets, and fluorescently labeled FFAs may not accurately reflect
the transport and metabolism of native FFAs [22,23]. As a result, the mechanism that transports FFAs across a cell membrane remains uncertain
(for recent reviews see [24-27]). Using quantitative mass imaging with MIMS we have directly studied the accumulation
of 13C in cultured adipocytes incubated with 13C-labeled oleic acid (13C-OA; see [28] for further details). We measured a high level of 13C accumulation in intracellular lipid droplets. Quantitative MIMS images were obtained
in parallel for 12C-, 13C-, 12C14N-, and the isobars 13C14N- and 12C15N-. The relative excess of 13C was measured at three different locations: outside the cell, inside the cell but
outside the lipid droplets, and inside the lipid droplets.

The quantitative mass images of an adipocyte exposed to 13C-OA for 20 minutes are shown in Figure 4a–i. Images of the 12C-, 13C-, 12C14N- and 12C15N- ions or of the 12C-, 13C-, 12C14N- and 13C14N- ions were acquired in parallel. Images of the 12C- and 12C14N- ions (Figure 4a,d) show the cell histology. The mass image of the 13C- ion (Figure 4b) is similar to the 12C- ion image (Figure 4a) in form, but has a lower count rate. The pixel counts of the 13C- image include both the natural 13C and the supplementary 13C from the 13C-OA transported into the cell. This supplementary 13C is at a maximum at the intracellular lipid droplets, where FFAs accumulate. The
mass image of the internal control 12C15N- ions (Figure 4e) is similar to the 12C14N- mass image, yet with a much lower count rate. Each pixel of the sample resulting in
the 12C15N- image contains the fraction of 15N related to the 14N content by the natural ratio of 15N/14N. The enhanced contrast observed in the 13C-/12C- image (Figure 4c) is due to the excess 13C incorporated into the lipid droplets from the transported 13C-OA. The internal control 12C15N-/12C14N- ratio image (Figure 4f) has no contrast because in the absence of added 15N, the value of the ratio of 15N/14N is equivalent to the natural ratio across the analyzed field.

Figure 4. Fatty-acid transport in cultured adipocytes. (a-i) MIMS mass images of cells dried with argon after unwashed 3T3F442A adipocytes were
incubated with 13C- oleate. Images show (a) 12C-, (b) 13C-, (d) 12C14N-, and (e) 12C15N-, and their respective ratio images of (c) 13C-/12C- and (f) 12C15N-/12C14N-. (g) HSI image of the 13C-/12C- ratio (the numerator has been multiplied by 100); (h) an RDIC image of the same cells
before analysis with MIMS. RDIC images (500×) were obtained using a Nikon Eclipse
E800 upright microscope. (i) The 13C14N-/12C14N- distribution also reveals the excess 13C in the lipid droplets. O, outside the cells; I, inside but not in visible lipid
droplets; LD, inside the lipid droplets. The MIMS images are 60 × 60 μm (256 × 256
pixels) and were acquired in 40 min. (j) HSI of the 13C/12C ratio after 'shaving' (see text) the adipocyte shown in (a-i); the adipocyte had
been exposed to a high primary-ion beam current approximately 1,000-fold more intense
than for the previous analysis to quickly remove material from the sample surface
in order to analyze deeper within the cell. Field: 60 × 60 μm (256 × 256 pixels);
acquisition time 10 msec/pixel. (k) Bar graph of the mean and standard deviation values of the 13C-/12C- ratio in 3T3F442A adipocytes. O, outside the cells; I, inside but not in visible lipid
droplets; LD, inside the lipid droplets. 13C-/12C- ratio values are shown after subtraction of the natural abundance ratio (1.2%). Adapted
with permission from [28].

The HSI image of the 13C-/12C- ratio is shown in Figure 4g, and the same cell photographed by RDIC microscopy on the silicon chip before analysis
with MIMS is shown in Figure 4h. The 13C-/12C- ratio, indirectly measured as the cyanide ion, 13C14N-/12C14N-, is shown in Figure 4i; this also shows accumulation of 13C- in the droplets. In contrast to the high 13C-/12C- ratios found in cells that were incubated with 13C-OA, cells washed with buffer solution after 13C-OA incubation had low 13C-/12C- ratios (images not shown). In cells not treated with 13C-OA, the value of the 13C-/12C- ratio measured under the same conditions was 1.15 ± 0.10%, not significantly different
from the terrestrial 13C/12C value of 1.12%. An indication of the accuracy of these values was obtained from
measurements of the 12C15N-/12C14N- ratio, whose value was 0.36 ± 0.01% in both washed and unwashed cells, in excellent
agreement with the natural abundance of 0.37%. The cumulative values obtained from
quantitative MIMS atomic mass images and extracted from the isotope ratio images are
shown in Figure 4k.

We can remove material quickly from the sample surface in order to study a variety
of depths within the cell. We refer to this as 'shaving' the sample. It is accomplished
in conditions that give a high primary-ion beam current (such as by removing the objective
diaphragm; see Figure 13 in the Discussion section). The results of such shaving are
shown in Figure 4j. The adipocyte analyzed in Figure 4a–i was shaved using, for a few minutes, a primary beam current approximately 1,000-fold
more intense than for the previous analysis. This uncovered a lipid droplet deeper
in the cell with a very high 13C-/12C- ratio, as shown in the HSI image (Figure 4j). Finally, MIMS allows us to acquire hundreds of atomic mass image planes successively
from the same cell, opening the door to full three-dimensional volume rendering. We
have begun using this capability to study the distribution of 13C among the lipid droplets located within a single adipocyte after incubation with
13C-OA [29]. In conclusion, MIMS can be used to investigate lipid metabolism with high spatial
and quantitative resolution [28]. Unlike other techniques, MIMS allows us to trace and to measure the movement of
native FFAs at specific subcellular locations.

Quantitative labeling of prokaryotic cells with gaseous 15N

The ability to image and measure stable isotopes makes it easy and safe to apply MIMS
to samples labeled with a gaseous precursor. Here we describe the application of MIMS
to the study of nitrogen fixation in bacteria (Figure 5a–k). Teredinibacter turnerae is a diazotrophic (nitrogen-fixing) marine bacterium that can be isolated from the
tissues of wood-boring marine bivalves (family Teredinidae) and grown in pure culture
[30,31]. Enterococcus faecalis is a bacterium that does not fix nitrogen. Both were cultured for 120 hours in a 15N atmosphere. Mass images of the 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. T. turnerae is barely visible at mass 12C14N- (Figure 5a) but is seen as intensely labeled at mass 12C15N- (Figure 5b) because it has used gaseous 15N to build its molecular constituents. By contrast, E. faecalis is visible at mass 12C14N- (Figure 5a) but not at mass 12C15N- (Figure 5b) because it does not use gaseous nitrogen and therefore has not incorporated 15N above the natural ratio. The HSI image of the 12C15N-/12C14N- ratio (Figure 5c) shows E. faecalis with an isotope ratio equivalent to the natural isotope ratio (blue) and T. turnerae, which has incorporated an enormous quantity of 15N, with an isotope ratio at least 100 times higher (magenta).

MIMS can also be used to study the distribution of isotope tag incorporation within
a bacterial population. The heterogeneity of nitrogen fixation among a population
of T. turnerae is shown in Figure 5d, where the same field cultured for 32 hours in a 15N atmosphere is shown as a series of HSI 12C15N-/12C14N- ratio images. The different HSI panels reveal the level of 15N incorporation in bacteria using a compressed color scale as described in the legend
to Figure 5. This analysis reveals the location and the distribution of ratio values, in other
words of nitrogen fixation, among the bacteria within the analyzed field. Large differences
in the amounts of 15N incorporation by T. turnerae cultured for 96 hours in a 15N atmosphere are demonstrated by the HSI images of the 12C15N-/12C14N- ratio (Figure 5e,f). Differences are visible among a few bacteria in contact with each other (Figure
5e) and even within a single bacterium (Figure 5f).

MIMS can detect and measure the function of intracellular bacteria within eukaryotic
cells. This is shown by the quantitative imaging of the incorporation of 15N in T. turnerae living in the gill bacteriocytes of a shipworm (Lyrodus pedicellatus) raised under a 15N atmosphere, as shown in the HSI image of the 12C15N-/12C14N- ratio (Figure 5g). The bacteria in the bacteriocytes that have incorporated 15N are shown in colors between yellow and magenta (the shipworm tissue is blue). An
internal control is the isotope ratio 13C-/12C- of the same field (Figure 5h), which shows a lack of contrast. The uniformity of the carbon ratio image eliminates
the possibility of artifacts in the nitrogen ratio image as a result of morphologically
or instrumentally induced isotope fractionation.

These quantitative images show that the MIMS method will be a powerful tool in the
investigation of nitrogen fixation. It can also be used to study bacteria in natural
environments and to explore the activity of diazotrophic symbionts in the tissues
of plants and animals. It is worth noting that the size of an object can be estimated
directly from the pixel signal intensity. This is illustrated with the flagellum visible
on one T. turnerae cell (Figure 5i). For example, at mass 12C15N (Figure 5j), we have a mean of 1,473 counts per pixel on the bacterium (Figure 5k, inset, red box). A line profile of the counts per pixel across the flagellum of
T. turnerae at mass 12C15N is shown in Figure 5k. The pixel crossed by the flagellum registered 66 counts. All conditions being approximately
the same, the number of counts is proportional to the surface area of the material
sampled in one pixel. In this particular image of 60 × 60 μm, 256 × 256 pixels, one
pixel is equivalent to 234 nm covering an area of 54,756 nm2. If the length of the flagellum crosses a pixel, 66 counts would represent a width
of (54,756 nm2/1,473 counts) × (66 counts/234 nm) = 10.5 nm; using the count values for the same
pixels at mass 12C14N, the estimate is 10.6 nm, which is approximately the diameter of a T. turnerae flagellum.

Use of double labeling with bromodeoxyuridine and 15N-leucine to measure protein renewal

Because MIMS analysis sputters only a few atomic layers, a sample can be reanalyzed
many times. This is illustrated by double-labeling studies of protein renewal and
DNA replication in the mouse kidney after ischemia. We have previously shown [32] in the mouse that 30 minutes of bilateral renal ischemia, resulting in significant
increases of blood urea nitrogen and creatinine, leads to protection of the mouse
kidney against a subsequent ischemic insult 8 or 15 days later, even when the second
ischemic period is extended to 35 minutes. Graded levels of time of initial ischemia
resulted in graded levels of protection 8 days later. Bromodeoxyuridine (BrdU) and
15N-leucine were administered to mice subsequent to the first ischemia, in order to
characterize the different response in cell proliferation in preconditioned and non-preconditioned
kidneys exposed to ischemia on day 8 after the initial surgery. Quantitative MIMS
images were recorded from thin sections of epon-embedded kidneys. The images were
acquired in parallel at mass 12C14N to show a morphological overview, at mass 31P to view the cell nuclei and at mass 81Br to identify the nuclei undergoing DNA replication, as shown by BrdU incorporation.
Protein renewal was calculated from parallel imaging at mass 12C14N and mass 12C15N.

A first MIMS analysis of a 100 × 100 μm field for 2 minutes, as shown in Figure 6a,b, reveals that one nucleus (in the 81Br- image in Figure 6b) has replicated its DNA, whereas the others have not. A second MIMS analysis at higher
resolution of cells around this replicating nucleus is shown in Figure 6c–e. The replicating nucleus is seen in the 81Br- mass image (Figure 6e), and the 31P- image (Figure 6d) shows the presence of two other nuclei that did not replicate. A third MIMS analysis
was performed on the same field at masses 12C14N (Figure 6f) and 12C15N (Figure 6g) to quantitate the protein renewal and compare renewal between replicating and non-replicating
cells. We found that incorporation of 15N into the replicating nuclei was twice as high as that in either the cytoplasm or
in non-replicating cells (nuclei or cytoplasm; Figure 6h). The 12C14N images acquired successively (Figure 6c,f) show that there are no visible changes; this validates the comparison of data obtained
in the second and third analysis. MIMS can thus be used with multiple tags that can
be studied with a succession of parallel analyses of the same field at a variety of
isotope combinations.

Qualitative labeling with stable isotopes

MIMS methodology enables us to study the spatial aspects of metabolic pathways and
the spatial relationship between replication and transcription. As an example, we
will show MIMS atomic mass imaging of the co-localization of RNA and DNA. Rat embryo
fibroblasts were pulsed with 15N-uridine and BrdU, markers of newly synthesized RNA and DNA, respectively. The simultaneously
recorded distributions of 12C15N- and 81Br- are shown in Figure 7a,b. As expected, the bromine signal (DNA) is restricted to the cell nuclei; there is
strong 81Br labeling along the nuclear envelope and around the nucleoli (Figure 7b). In contrast, the 12C15N- signal (RNA) is strong within the nucleoli and along the nuclear envelope (Figure
7a).

Figure 7. Qualitative co-localization of DNA and RNA through simultaneous imaging of RNA and
DNA. Rat embryo fibroblasts were pulsed with 15N-uridine and BrdU as markers of newly synthesized RNA and DNA, respectively. (a,b) Parallel mass images at (a) 12C15N- and (b) 81Br-. (c) Overlay of 12C15N- and 81Br- images. 12C15N- is depicted as red (R) and 81Br- as green (G); the overlap between them shows up as yellow. (d) Overlay of 12C14N- and 12C15N- images. 12C14N- is depicted as red (R) and 12C15N- as green (G); the overlap between them shows up as yellow. Conditions of MIMS analysis:
beam current 2pA; beam diameter 100 nm; field 20 × 20 μm.

An overlay of the 12C15N- signal in red with the 81Br- signal in green shows the co-localization of newly synthesized RNA and DNA in yellow
(Figure 7c). This co-localization, visualized directly from the isotope images, avoids the complications
potentially introduced by immunochemical methods [33]. Localization of newly synthesized RNA requires distinguishing a local excess of
15N over its natural occurrence. We carried out another MIMS analysis of the same cells
to record the distributions of 12C14N- and 12C15N- in parallel (in our prototype instrument, we cannot simultaneously detect the isobars
12C14N- and 12C15N- together with 81Br- because of the steric hindrance of the electron multipliers). Overlaying the 12C14N- signal in red with the 12C15N- signal in green shows up the local excesses of 15N above its natural occurrence in yellow (Figure 7d). The yellow identifies the localization of 15N-uridine-labeled newly synthesized RNA within the nucleoli, along the nuclear envelope,
and in the cytoplasm of the top cell.

The importance of parallel detection and isotope ratio imaging is illustrated by the
following fortuitous observation. We used MIMS to study the distribution of RNA in
the nucleolus by studying fibroblasts cultured in the presence of 15 N-uridine. Quantitative mass images of 12C-, 12C14N- and 12C15N- secondary ions were acquired in parallel. In the fibroblast shown in Figure 8, the nuclear membrane is clearly visible at mass 12C14N- (Figure 8a), and two nucleoli are seen highly contrasted at masses 12C14N- and 12C15N- (Figure 8a,b). Higher resolution parallel mass images of the nucleolus seen on the right in Figure
8a are shown in Figure 8c–e. In these cells embedded in epon – a polymer lacking nitrogen – the 12C- image (Figure 8c) shows little contrast except for a dark spot (diameter around 122 nm) in the middle
(Figure 8c, red arrow; the low brightness indicates low counts). This spot was caused by accidental
exposure of the sample to an intense stationary primary-ion beam. The spot is also
seen in the 12C14N- and the 12C15N- images (Figure 8d,e). The 12C15N- image, however, contains four additional dark regions (Figure 8e, white arrows), ranging from 200 to 280 nm in diameter. Nevertheless, isotope ratio
and HSI derivation of 12C15N- and 12C14N- images clearly distinguish the accidental dark spot, which has a high level of 15N incorporation, from the other four sub-nucleolar regions, which have low 15N incorporation; they can therefore be taken to be related to nucleolar organization
(Figure 8f,g). We also used MIMS to evaluate the dose response of uridine incorporation in nucleoli
of rat embryo fibroblasts cultured in the presence of 0.0, 0.01, 0.1, and 1.0 mM 15N-uridine (Figure 8h), demonstrating that MIMS may be used to establish a dose-response curve at the level
of intracellular organelles.

Figure 8. Distinguishing between an artifact and the subnucleolar heterogeneity of 15N-uridine incorporation. (a,b) Parallel quantitative mass images of (a) 12C14N- and (b) 12C15N- images of a fibroblast cultured in the presence of 15N-uridine. Ncl, nucleoli; NM, nuclear membrane. Field: 40 × 40 μm (image has been
cropped); acquisition time 20 min. (c-e) High-resolution parallel mass images at 12C-, 12C14N- and 12C15N- of the large nucleolus seen in (a,b). Field: 8 × 8 μm; acquisition time 30 min. (c)
12C- image, arising from both tissue and embedding medium; the dark spot (red arrow) was
caused by accidental exposure to a stationary high-intensity primary Cs+ ion beam. (d) 12C14N- image. (e) 12C15N- image, showing subnucleolar areas of low local 15N incorporation (white arrows). (f) Ratio of the (d) 12C14N- and (e) 12C15N- images; here, the 'dark spot' (red circle) is barely visible because the value of
the 12C15N-/12C15N- ratio is close to that of the surrounding area. (g) HSI image of the 12C15N-/12C14N- ratio (the numerator has been multiplied by 10,000). The 'dark spot' isotope ratio
is close to that of the surrounding area. Subnucleolar regions of low incorporation
of 15N-uridine stand out in both the (f) ratio and the (g) HSI images. (h) Calibration with 15N-uridine. The graph shows the intranucleolar accumulation of 15N-uridine (measured as 12C15N-/12C14N- (experimental – control)/control) as a function of the concentration of 15N-uridine in the culture medium.

Use of MIMS without isotope labeling to study gross differences in subcellular composition

Quantitative mass images of the chemical elements within a cell can provide information
on the existence and location of subcellular domains with gross differences in composition.
Thus, even without exposing the cells or tissues to isotopically labeled molecules,
we may obtain a measure of the gross cellular composition at the level of microdomains
that cover areas of sub-micrometer size. This is illustrated by the overlay image
of an endothelial cell analyzed in parallel for 12C-, 12C14N- and 31P- (Figure 9). Striking differences in gross composition within a cell are revealed. The area
over the nucleus and the thicker part of the cytoplasm is intensely red, an indication
of comparatively high nitrogen content; the wide area at the periphery (the lamellipodium)
is relatively rich in phosphorus and poor in nitrogen; and at the outmost edge of
the cell, there is relatively more carbon than in the wide part of the lamellipodium.
Short, thin protrusions with a relatively high nitrogen signal can also be seen at
the very edge of the cells; these are probably filopodia. One may assume the following:
high 12C14N indicates protein (or glycoproteins); high 12C14N associated with phosphorus indicates nucleotides; 12C with less 12C14N indicates lipids or sugars; and 12C associated with 31P indicates phospholipids.

Figure 9. Analysis of gross differences in composition within an unlabeled cell. Endothelial
cells were cultured on silicon supports, fixed on the support, dried, and analyzed
with MIMS. Quantitative mass images of the surface of a whole endothelial cell were
recorded in parallel at masses 12C-, 12C14N- and 31P-. An overlay of these images is shown, with 12C14N in red, 12C in green, and 31P in blue. Scale bar = 10 μm.

We undertook a more detailed analysis of the region at the edge of the lamellipodia
of endothelial cells (see Figure 9), in which we saw domains two pixels wide (the pixel size here is 234 nm) containing
five times more nitrogen and a tenth the level of oxygen than the neighboring pixels.
From the original MIMS images acquired in parallel at masses 12C14N-, 12C- and 16O- (Figure 10a–c), together with HSI images of the ratio 12C14N-/12C- (Figure 10d) and of the ratio 16O-/12C14N- (not shown), we observe lamellipodial domains at the edge of the cell that look like
regularly spaced 'dots'. These are rich in 12C14N and poor in 16O compared with their surroundings. The values of 12C14N- counts, 12C- counts and of the 12C14N-/12C- ratio are shown in Figure 10f for a group of pixels constituting the central dot of the inset in Figure 10e, and the values of the 16O-/12C14N- and of the 12C14N-/12C- ratios are shown in Figure 10g for the same pixels; these panels illustrate the way in which quantitation and imaging
are intimately associated in MIMS. The white rectangle in Figure 10f,g surrounds two neighboring pixels that have the highest nitrogen content and the lowest
oxygen content (Figure 10h) compared with the surrounding lamellipodium (Figure 10i). Thus, parallel quantitative mass imaging using MIMS without isotopic supplementation
can identify nanometer-sized structures that may be functionally significant.

Figure 10. A detailed analysis of the edge of a lamellipodium of an unlabeled endothelial cell.
This example illustrates analysis by counts per pixel and HSI. (a-c) Three MIMS images acquired in parallel at (a) 12C14N-, (b) 12C-, and (c) 16O-. Field: 60 × 60 μm (256 × 256 pixels); acquisition time 2 min. (d) HSI image of the ratio 12C14N-/12C- (the numerator has been multiplied by 10). Magenta dots (arrowed) indicating areas
of high relative 15N incorporation appear at the edge of the lamellipodium. Field: 60 × 60 μm (256 ×
256 pixels). (e) HSI images of the ratios 12C14N-/12C- (left) and 16O/12C14N- (right; the numerator has been multiplied by 10). The regularly spaced dots (arrowed)
can be seen at the edge of the lamellipodium. (f) At each pixel, arranged from top to bottom, are the values of the 12C14N- counts, the 12C- counts and the 12C14N-/12C- ratio (multiplied by 10) for the pixels shown in the inset in (e). (g) The corresponding values at each pixel, arranged from top to bottom, for the 12C14N-/12C- ratio values (multiplied by 10) and the 16O-/12C14N- ratio (multiplied by 10). (h,i) Bar graph of the mean count values of 12C-, 12C14N- and 16O- on (h) the dot at the periphery of the lamellipodia and (i) the edge of the lamellipodia.

Labeling with radioactive 14C

MIMS opens the world of stable isotopes to quantitative nanoautography. It is similar
in principle but much more powerful than autoradiography because it is precisely quantitative,
needs much shorter exposure times, can use the very large number of stable isotopes
that are naturally available, can easily use multiple labels, gives high lateral resolutions,
provides exceptional depth resolution and would be harmless for clinical use. MIMS
can also be used for high-resolution quantitative imaging of radioisotopes (such as
14C), and with high sensitivity, as shown in the pioneering work of Hindie et al. [10]. An example of parallel imaging at masses 14C- and 12C15N- of a whole fibroblast pulsed with serum and then with 14C-thymidine after serum deprivation is shown in Figure 11a,b, and the overlay of the 12C15N- and 14C- images in Figure 11c. The 12C15N- and 14C- images from a control sample with no added 14C are shown in Figure 11d,e. The 12C15N- mass image maps the whole fibroblast (Figure 11a). The 14C- atomic mass image, indicative of the 14C-thymidine in the DNA, is restricted to the nucleus (mean count within nucleus =
49.0, SD = 38.2, n pixels = 2,388, sum of counts = 116,955). The 14C signal is variable across the nucleus and is segregated into domains, reminiscent
of the results of Wei et al. [34]. The mean background 14C count outside the fibroblast is 0.6 (SD = 1.1, n pixels = 31,148, sum of counts = 19,873). The mean 14C count in the nuclear region of the control sample is 0.009 (SD = 0.128, n pixels = 2,875, sum of counts = 25). The mean 14C count outside the nucleus of the control sample is not significantly different,
with a value of 0.008 (SD = 0.153, n pixels = 50,842, sum of counts = 420). Thus after exposure to 19 nmol/ml of 14C-thymidine, counts in the nucleus of the labeled sample have increased by a factor
of 6,125 relative to the control sample. Control background count of 14C is negligible. Compared with autoradiography, one can estimate that even if only
1‰ of the 14C atoms were sputtered and only 1% of the sputtered atoms were ionized, the sensitivity
of MIMS is at least 103-fold greater (see Additional data file 5 for calculations).

Use of MIMS to track individual cells in large cell populations

Following the transplantation of allogeneic tissues and organs, some donor-derived
cells are known to leave the graft and present alloantigens to the host's immune system
[35-37]. The exact nature and frequency of the donor cells infiltrating the host's lymphoid
tissues are still unclear, however. The movement patterns of recipient immuno-competent
cells that recognize the alloantigens are also unknown. These questions have remained
unanswered because of the lack of sufficiently sensitive techniques for accurately
tracking and visualizing small numbers of lymphocytes in tissues. Here, we use MIMS
to track donor cells after transplantation.

Spleen cells from C57Bl/6 (B6, MHC haplotype H-2b) mice fed for 2 weeks with a 15N diet were injected subcutaneously in the footpad of a fully allogeneic BALB/c mouse
(H-2d). Two days later, popliteal lymph nodes (located behind the knee, where they drain
the foot pad of the mouse) were collected and examined by MIMS for the presence of
donor cells. Mass images of the 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. Because only a very few donor cells are expected to
be found among the recipient cells, a large number of cells need to be examined relatively
quickly with some means of highlighting the areas that could contain donor cell(s).
This was done using MIMS to rapidly map a large area of lymph node as shown in Figure
12a,b. We clearly observe areas where there appears to be a small excess of 15N above its natural abundance (Figure 12b). These areas were then reanalyzed with MIMS at a higher resolution over a longer
time. A field containing donor cells is shown in Figure 12c–e. In the 12C15N-/12C14N- ratio image (Figure 12d), two donor cells are clearly visible. The ratio image of 13C-/12C- of the same field taken simultaneously (Figure 12e) does not show any contrast and acts as an internal control. The frequency of transplanted
cells infiltrating the recipient lymph node was estimated to be 690 cells per million.

We conclude that the MIMS technique allows the detection and frequency determination
of rare cells infiltrating the recipient's lymphoid tissues after transplantation.
The methodology can be extended to study solid organ transplants and the nesting of
stem cells. It is important to note that the current techniques for tracking transplanted
cells are fraught with difficulties [38]. Labeling DNA with stable isotopes should provide a method allowing long-term and
physiological marking, the possibility of determining the generation of single cells
from the amount of label within the cell, and the possibility of labeling both donor
and recipient cells differently, enabling the unambiguous recognition of cell fusion
or phagocytosis of donor cells by the recipient's cells [38].

Discussion

We have shown that we can obtain quantitative atomic mass images using MIMS. Because
these images are produced in parallel from the same sputtered volume they are in exact
register with each other, which is necessary to derive meaningful isotope ratios.
A lateral resolution that reaches 33 nm, in theory, and a high mass resolution (a
mass/change in mass ratio of approximately 10,000) at high secondary-ion transmission
(70–80%) allows us to image and quantitate molecules labeled with stable (or radioactive)
isotopes within subcellular compartments. The high mass resolution allows us to distinguish
the distribution of isobaric atomic species. For example, by separating 12C15N- from 13C14N- and 13C- from 12C1H-, we can image the intracellular distribution of molecules labeled with the stable
isotopes 13C and 15N (such as 15N-thymidine and 13 C-uridine) in parallel and in the same preparation. Finally, the high stability of
the primary beam, the mass spectrometer and the detectors contribute to precise quantification.
These unique characteristics have allowed us to develop MIMS.

Instrumental advances

Instrument modifications

In SIMS, a primary-ion beam (usually Cs+ or O-) is used as a probe to scan across the surface of a solid sample. The impact of a
primary ion on the surface of the sample triggers a cascade of atomic collisions,
and atoms and clusters of atoms are ejected, most originating in the neighborhood
of the impact point. In the ejection process, some of them will be spontaneously ionized;
these secondary ions are characteristic of the composition of the target area (see
Figure 1). After separating the secondary ions according to their masses, an image of the
surface composition in respect of a selected mass can be recorded. We used a new generation
of SIMS instrument that can detect several secondary ions at once; this and several
other advances detailed below represent major improvements over previous generations
of SIMS. The machine is the prototype of the NanoSims50 built at ONERA (Office National
d'Études et de Recherches Aérospatiales) jointly with UPS (Université Paris-Sud) and
upgraded by Cameca, Courbevoie, France, and is shown in Figure 13.

Figure 13. Diagram of the prototype NanoSims50 used in MIMS. The main components of the instrument
include: the primary column (PC) with a cesium primary-ion source (IS) used to sputter
the surface of the sample and to enhance the yield of negative secondary ions and
a series of lenses (L0, L1, L2 and L3) to shape the primary beam; the objective column
(OC), where the same ion optic is used in a coaxial manner both to focus the primary
beam on the sample (S) and to collect the secondary ions (the primary-ion beam is
focused on the sample with the objective lens (EOP) and aperture limited with the
diaphragm (D1), the secondary ions are collected with the secondary-ion focusing lens
(EOS)); the secondary column (SC) where the secondary-ion beam is shaped to match
the acceptance of the spectrometer (the secondary column contains an entrance slit
(ES), a corrector (CY) and deflectors (P2 and P3) to center the secondary-ion beam
in the entrance slit); an aperture slit (AS) to reduce the angular aberration of the
secondary-ion beam; and the mass spectrometer, made up of the association of an electrostatic
prism (EP) and a magnetic prism (MP), which enables the focusing of the secondary
ions as narrow lines along the focal plane (FP) of the magnet. Chromatic aberrations
are minimized with the quadrupole (Q) and the slit lens (LF4). Energy aberration is
reduced with the energy slit (WS). The transmission at high mass resolution is improved
by correcting the main second-order aperture aberrations with the hexapole (HX) placed
in front of the electrostatic prism, tilting the entry and exit faces of the magnetic
prism, and focusing in the vertical section using additional lenses placed between
the electrostatic and magnetic prisms. The deflectors (C4) help to center the masses
in the detectors. In the multi-collection chamber (MC), four detectors can be moved
along the focal plane. Each detector is made up of deflection plates (DP) followed
by a selection slit (SS) and a miniature electron multiplier (EM). The deflection
plates, which permit scanning of a small portion of the spectrum, greatly improve
the final tuning of mass lines. This instrument provides both parallel detection and
high mass resolution with little sacrifice in secondary-ion transmission from sample
to detector. As a particularly striking example, in Figure 15, the 12C- secondary ions were detected at 90% relative transmission with a mass resolution of
1 part in 11,825.

The objective column (OC) in the new SIMS instrument (see Figure 13), where the primary ions and secondary ions share the same path but in opposite directions,
is a big step forward. It allows for a very short focal length for the ion-beam focusing
lens (EOP) and for the secondary ion extracting lens (EOS) leading to a significant
reduction in various aberrations. In addition, the incidence of the coaxial primary-ion
and secondary-ion beams normal to each other allows us to reduce the geometrical dimensions
of the instrument, leading to a significant reduction in various aberrations. The
improvement rests on using the same lens for focusing the primary-ion beam, and for
collecting the secondary ions that are formed. Secondary ions are emitted at a wide
range of angles and energies. To collect them efficiently a strong electrostatic accelerating
field is needed near the sample surface to gather their trajectories close to the
surface normal. The conjugate action of the immersion lens and other lenses in the
secondary optical column gives the secondary ion beam springing from the primary ion
probe impact area the appropriate shape for matching the acceptance of the mass spectrometer
tuned for high mass resolution. It should be emphasized that getting high mass resolution
and high collection efficiency together is possible because the impact area of the
primary-ion beam is very small (less than 0.1 μm2). This allows us to keep the beam small during its transport from the sample to the
entrance of the spectrometer. From the general laws of optics, however, we can do
this only for one position of the beam on the surface. As soon as the primary-ion
beam moves, the secondary-ion beam emitted from the impact area is displaced as a
whole in front of the spectrometer and a variable fraction of the beam (or no beam
at all), depending upon the amplitude of the motion, enters the spectrometer.

To solve this problem, the machine uses an operation mode called 'dynamic transfer'
to keep the secondary-ion beam stationary at the entrance of the spectrometer while
the probe scans the surface. Dynamic transfer is achieved by the set of deviation
plates, which move in synchrony with the scanning primary-ion beam. Because of the
coaxial layout, those plates move both secondary and primary beams, but whereas they
act on the secondary beam to align it along the optical axis, they act on the primary
beam to make it rotate about the center of the aperture stop (see D1, Figure 13), which limits aperture aberrations on the probe. As a result, not only does the
secondary beam stay stationary, which ensures a constant collection efficiency over
the scanned area, but the intensity of the primary beam remains constant during the
scanning; both of these avoid the 'vignetting', which is a gradual darkening of the
corners of the ion images as the size of the field of view increases.

High mass resolution is obtained because the initial energy of ion spread has been
cancelled at the level of the mass lines by coupling the electrostatic and magnetic
prisms via a quadrupole lens. In addition, the elaborate ion optics allows the substantial
reduction of second-order aperture aberrations, which improves the mass-resolving
power (for example, at mass 27 the separation of 12C15N from 13C14N). This results from both the suppression of second-order aperture aberrations of
the magnetic prism by tilting the entrance face of the magnetic prism, and the reduction
of second-order aberrations of the electrostatic prism by placing a hexapole in front
of the electrostatic prism (see Figure 13).

The mass spectrum of the secondary ions is displayed along the focal plane as a set
of well-resolved mass lines. It is a discrete spectrum with wider spacing between
ions separated by 1 mass unit and closer spacing when multiplets (isobaric species)
occur. Because of the limited size of the magnet, however, the whole spectrum cannot
be displayed at once. The ratio between the lower and the higher masses in the displayed
range is about 20. By changing the strength of the magnetic field it is possible to
choose the lower mass and, in consequence, the higher one. For instance, if 12 is
the lower mass unit, the spectrum is displayed up to mass number 240.

Mass selection for discriminating the secondary ions is provided by individual detector
'trolleys' in the mass spectrometer, each containing a set of deflection plates followed
by a selection slit and an electron multiplier (see Figure 13), which are moved along the focal plane to the appropriate positions corresponding
to the masses of interest. The positions depend on the kinetic energy of the secondary
ions and the magnetic field strength of the mass spectrometer. For example, in the
analytical run shown in Figure 14, one trolley was positioned for detecting atomic mass 12, a second for mass 13, a
third for mass 26, and a fourth for mass 27. As stated earlier, the ability to detect
several masses simultaneously at high mass resolution is novel, and is essential to
derive meaningful isotope ratios from a sub-cubic-micrometer sample volume. Thus,
in the example shown in Figure 14, it is necessary to distinguish at mass number 27 between 12C15N- and 13C14N-. This last level of mass selection is conveniently obtained by operating the deflection
plates in front of the selection slit.

Figure 14. Parallel quantitative mass imaging with MIMS, using a cochlear field as an example.
A schematic representation of the MIMS instrument is shown at the top, with the secondary
ions beings selected in parallel by four detectors (T1–T4) which are moved along the
focal plane to the appropriate positions for a given mass. The 12C-, 13C-, 12C14N- and 12C15N- quantitative mass images recorded by the four detectors are shown below.

Recordings from individual electron multipliers when the deflection plate voltages
are adjusted gradually so as to scan across a narrow range of secondary-ion masses
are shown in Figure 15. By setting the voltages appropriately it is possible to scan finely the multiplet
of the already highly resolved mass lines and to determine which voltage is suitable
for isolating the mass of interest, practically eliminating any spillover from neighboring
mass peaks. It should be noted that the width of the mass selection slit is larger
than the mass line itself. This configuration allows us to make precise isotopic measurements
because it allows for small drifts of the mass line that can result from uncontrolled
instrumental factors. As a consequence, the mass line appears in the recordings as
a peak with a flat top (Figure 15). Two adjacent mass lines can be completely separated and appear in the recording
with no valley between them (Figure 15b–d and Additional data file 6). It can be seen from Figure 15b–d that different isobars with very similar masses can be distinguished, such as 13C- and 12C1H- (mass 13.003355 and mass 13.007825, respectively; Figure 15b); 12C14N- and 1H12C13C- (mass 26.003074 and mass 26.011180, respectively; Figure 15c); 12C15N-, 13C14N- and 12C14N1H- (mass 27.000109, mass 27.006429 and mass 27.010899, respectively; Figure 15d).

Figure 15. High-resolution mass spectra illustrate the resolving power of our prototype NanoSims
instrument. The detectors are positioned along the focal plane at the focal points
for the atomic masses (a) 12, (b) 13, (c) 26, and (d) 27 as shown in Figure 14. Varying the electrical potential of the deflector plates
selects between different isobaric ions. Note that with the log scale and the counting
software, when zero counts are recorded, the data point is recorded as 1/T, where T is the counting time in seconds for each data point. In (a-c) the counting time was
0.5 sec, and the background count rate appears as 2 counts/sec even though it is zero
counts; in (d), the counting time was 10 sec and a background count rate of zero is
not visible. See Additional data file 6 for more details on the shape of the peaks.

A single ion impact on the first dynode of an electron multiplier ejects several secondary
electrons, which are subsequently amplified by the other dynodes in a chain amplification
process and generate a voltage pulse. Pulse heights depend upon the ion-electron conversion
yield on the first dynode and upon the voltages applied on other dynodes. Pulse heights
have a distribution that can be stretched by changing the high voltage. The distribution
is usually depleted on the low-amplitude side, where one must account for the superposition
with low-amplitude pulses from the electronic noise. A pulse-height discriminator
eliminates low-amplitude contributions so as to select only signal pulses. Of course,
a fraction of the signal is suppressed in that process, but this fraction is quite
small, for example a few percent for CN-. The secondary-ion detection efficiency is set to be equivalent among the detectors
in part by adjusting the rejection threshold and in part by adjusting the high voltage.
Measurement of samples of known isotopic composition can be used to correct for any
remaining differences in efficiency between detectors. Using the adjustments and tuning
described above, we can measure the isotope ratios of secondary ions, which is at
the core of MIMS methodology, in a precise and reproducible manner.

Spatial resolution

The parallel detection of different ions, the small size of the primary-ion beam,
and the high efficiency of secondary-ion collection all contribute to the highly effective
spatial resolution of MIMS, reaching 33 nm, 30-fold better than in previous instruments.
Smaller beams are possible and may be useful for locating and measuring highly concentrated
tags in volumes of the order of (20 nm)3. Primary sources with higher brightness could give smaller probes with sufficient
current for practical analysis: they could be provided by a brighter Cs+ primary-ion source or by other types of sources [4].

Volume sputtered and detectability limit

The absolute sensitivity of SIMS is among the highest of current chemical analysis
methods and the total amount of material needed for MIMS analysis is extremely small.
Using MIMS, one can detect parts per billion of an isotope in a sub-cubic-micrometer
volume of sample. Let us assume an analyzed field of 6 × 6 μm, a primary Cs+ beam current intensity of about 0.4 pA, a dwell time per pixel of 20 msecs, 256 ×
256 pixels, a sputtering efficiency of five target atoms per Cs+ ion, an atomic layer thickness of 2.55 × 10-4 μm, and an atomic density of 6 × 1010 atoms per μm3: the total sputtered volume is then 0.27 μm3, and the sputtering rate is 0.4 nm/minute or 1.5 atomic layers/minute. For a probability
of detection of 95%, three ions have to be emitted; assuming a useful ionization yield
of 1 × 10-2 ions/atom, then the minimum detectable amount is 1.85 × 10-8 or approximately 20 parts per billion (see Additional data file 1).

Contrast formation

There is no theory using first principles that allows us to predict what happens when
the surface of the sample is hit by a primary ion. Which atoms are most likely to
escape?

What is the sputtering rate (the rate of removal of target atoms)? When a given atomic
species is sputtered, what is the ionization yield (the fraction of atoms that are
ionized)? How will the composition of the matrix affect the number of atoms sputtered,
the ionization yield, and the formation of polyatomic clusters such as CN or CNH?
What is the breaking pattern of large and small molecules in atoms and atomic clusters?
How interdependent are the parameters mentioned? All these questions are yet to be
answered. In addition, the choice of primary ions, so important in practice, remains
mostly empirical. Indeed, in order to achieve the high ion yields required for trace
analysis down to the low parts per billion range, the sample needs to be bombarded
with or exposed to chemically reactive species, and O- or Cs+ are most commonly used. There is, however, a lack of detailed knowledge about the
instantaneous surface composition, and all attempts to correlate observed ion yields
with available ionization models should be considered highly questionable [39].

The contrast of a mass image is due to regional differences in secondary-ion currents
emitted over the analyzed field. The main determinants of secondary-ion formation
are the sputtering rate (the rate of removal of the target atom by the primary ions),
the ionization yield (the fraction of sputtered atoms that are ionized) and the local
concentration of atoms. These parameters cannot be derived directly from first principles,
but we may safely assert the following. The sputtering rate in steady state is directly
proportional to the primary beam current density at the sample surface and is dependent
on the local atomic composition.

The ionization yield, in the negative secondary-ion emission mode, depends strongly
on both the electron affinity of the elements or clusters under consideration and
the concentration of the Cs+ ions that are implanted by the beam into the superficial atomic layers of the sample.
To a first approximation, implanted primary-ion concentrations vary as the inverse
of sputtering rates; this makes the ionization yield somewhat a function of the sputtering
rate. However, we do not know the form of the function that links implanted Cs+ concentration to ionization yield.

Taking all these factors in consideration, we can predict the following. A low sputtering
rate is generally associated with a higher concentration of implanted Cs+; lower sputtering rates should therefore lead to higher ionization yields. If ionization
yields are strongly dependent on Cs+ concentrations, low sputtering rates may paradoxically lead to higher secondary-ion
currents. In a situation in which the sputtering rates of different areas over the
field of analysis are not the same, a given area may appear brighter because the sputtering
rate is lower (with a relatively higher concentration of implanted Cs+) and the ionization yield is still sensitive to the concentration of implanted Cs+. Moreover, the ionization yield is likely to depend on local sputtering rates because,
once a steady state has been reached, these local rates control the local concentrations
of the implanted primary ions. As a consequence, the brightness of an image area depends
upon the local concentration of the element under consideration, the implanted Cs+ concentration and the sputtering rate.

Without any contrast, images would not exist. Parallel mass images of different elements
with different electron affinities convey a lot of information. For instance, a section
of embedded cells bombarded with Cs+ gives 12C- images with low contrasts and, in parallel, 12C14N- images showing strong contrasts related to known cell structures (for instance, Figure
14, 12C- and 12C14N-). Let us first consider 12C- images. The local 12C- intensity depends on both the sputtering rate and the ionization yield. When analyzed
in parallel, one would expect (but it is not demonstrated) ionization yields of 12C14N- ions, with their high electron affinity (3.6 eV), to saturate (to reach their maximum
electron capture probability) more rapidly during Cs+ implantation than 12C- ions, whose electron affinity is much lower (1.2 eV). It follows that, if the ionization
yield is strongly dependent on the concentration of implanted Cs+, at low concentrations of implanted Cs+ the 12C- ion emission should be more sensitive to small changes in the implanted Cs+ and may be more sensitive than 12C14N- ions to local differences or fluctuations in sputtering rate.

The low contrast that we observe in 12C- images of epon-embedded samples probably indicates that sputtering rates are evenly
distributed across the field analyzed and that implanted Cs+ concentrations are approximately equivalent throughout the sample. The remaining contrasts
are probably due to a slightly lower concentration of carbon in the biological tissue
than in the embedding epon and to the formation of 12C14N atomic clusters that reduce the total number of 12C atoms available to form 12C- ions. If high contrasts were observed it would alert us to differences in sputtering
rates or unexpected differences in atomic concentrations of carbon.

Because 12C- images indicate a homogeneity in the sputtering rate and a steady state of the implanted
Cs+ concentration, the contrast observed on the CN- images acquired in parallel must reflect differences in local nitrogen concentration
over the analyzed field and cannot be due to emission artifacts. The fact that, in
many instances, interpretation of 12C14N- images is straightforward and allows us to recognize easily details of features in
tissue or cell sections is a boon.

Finally, we wish to emphasize the strength of the quantitative information derived
from the ratio of mass images acquired in parallel. All parameters being the same,
atoms or clusters such as 26CN and 27CN are affected identically at a level of precision well beyond what is needed for
our experiments, and the quantitative ratio images contain the true isotope ratio
values, cancelling out sputtering, ionization and other effects that affect the emission
of a single ion.

The relationship between contrast formation, lateral resolution and counting statistics

The lateral resolution is essentially a function of the size of the primary ion beam.
The smaller the beam diameter, the higher the resolution; to be visually appreciated,
however, the image has to have enough contrast; that is, the image has to contain
enough counts. Higher counting rates will be obtained with a brighter primary ion
beam, all other conditions being the same. The count rate does not affect the intrinsic
resolution, but it is a main determinant of the counting statistics. For example,
in a parallel set of 12C15N- and 12C14N- images, although a 12C15N- region in general contains far fewer counts than its 12C14N- homolog, it has the same lateral resolution. The variance of the 12C15N-/12C14N- isotope ratio, however, is essentially determined by the number of counts in the 12 C15N- region.

If the sample surface being studied is not flat but shows relief, we observe a three-dimensional
appearance in the sample image. Here, the contrast may be due either to a differential
implantation or to a differential sputtering that occurs because the primary beam
attacks the sample surface at a variety of angles. It should be emphasized, however,
that these phenomenon affect in the same proportion – at least to a first approximation
– isotopes separated by 1 atomic mass unit. Thus, the variations in count rates cancel
out (note that all relief effects disappear in the ratio images; see for example the
adipocyte in Figure 4d,f), and the value of the isotope ratio still represents the local abundance of the
individual isotopes with a precision of a few percent, which is entirely satisfactory
for biological purposes. SIMS had previously been used for quantitation in the absence
of imaging, and for imaging in the absence of quantitation. The advantage of MIMS
is to combine high spatial resolution (that is, analyzing a much smaller amount of
material than previously), with simultaneous (parallel) acquisition of information
from several atomic masses, and with isobar separation at mass 27.

The ability to provide quantitative information is a unique attribute of MIMS. Imaging
is a guide for exploiting the massive amount of unique quantitative information, which
is based on the mining of the counts of secondary ions per pixel.

Isotope ratios

The isotope ratio values are the essential data needed to interpret the results, and
the derivation of isotope ratios is an integral part of MIMS methodology. The absolute
number of counts for a given atomic mass do not convey any information about the presence
of an isotope excess. For example, the counts of 12C15N by themselves do not allow us to conclude anything about the presence of a 15N tag in the analyzed region. Only the ratio 15N/14N compared with the natural abundance of 15N is informative. The visual observation of contrast and/or brightness is no more
informative, and may be misleading. In a given field, contrast is due to variations
of the number of counts per pixel, which may have multiple undocumented causes. As
discussed under 'Contrast formation' earlier, in addition to the local abundance of
the isotope under consideration, the secondary-ion intensity depends on other parameters
such as the number of atoms of Cs implanted and the quality of the matrix into which
the isotope is introduced. Thus, the fact that a given area emits more than a neighboring
one does not mean that it contains more of the isotope tag. High counts in 12C15N do not mean that there is an excess of 15N until they are normalized with respect of the counts of 14N by calculating the isotope ratio12C15N/12C14N. A region of high secondary-ion yield at mass 12C14N may have little incorporation of 15N (see, for example, the cochlear outer pillar cells (Figure 3a), which have a high secondary 12C14N yield and very low incorporation of 15N (Figure 3c and Additional data file 3). Conversely, a region with high 15N on the 12C15N mass image compared with the neighboring pixels may simply reflect a region with
overall high yield of both 12C14N and 12C15N, even though their proportion is equivalent to the natural ratio. The isotope ratio
values for regions of interest are the only information that may be used for evaluating
where and how much a label such as 15N has been incorporated.

The power of the isotope ratio method is that all analytical parameters being the
same (field analyzed, instrumental conditions, Cs+ implantation, matrix effect), one may assume that they affect two isotopes of the
same atomic number – that is, isotopes that differ in general by 2 atomic mass units
at most – in an identical fractional manner, so that any unknown effect will cancel
out and that isotope ratio values will uniquely represent the relative abundance of
one isotope with respect to the other.

Ensuring 'flatness' of the field, such that the isotope ratio values among regions
of interest are equivalent regardless of their location in a control image field,
is very important. It demonstrates that there is no differential fractionation among
isobars: that is, that differences in isotope ratio among various areas are not the
consequence of changes in transmission between a set of isotopes that are related
to location within the field. This is why the 12C and 13C quantitative atomic mass images used to generate the 13C/12C ratio image (in Figure 3, for example) are an essential internal control. Because there is no 13C added, they control for the flatness of the field, which means: first, equivalence
of the 13C/12C ratio at any location over the field analyzed; second, an absence of instrumental
drift during analysis; third, the lack of some topographical effect; and fourth, the
lack of a differential matrix effect. With all conditions being the same and because
the secondary ions are detected in parallel from the same analyzed area, this ensures
that measured high values of 15N/14N ratios do show an excess of 15N and cannot be an artifact.

We have developed several kinds of controls. Two essential controls in our quantitative
imaging allow us to validate the analytical conditions. This validation is individual
to the field being analyzed. The first control is to make sure that if no isotope
has been added, the isotope ratio measured is equivalent to the natural abundance
in a region of the field where no excess isotope is expected. When the sample is an
epon-embedded section, this is measured on the epon, in a region of the analyzed field
that does not contain tissue or cells.

The second essential control is to make sure that, for every field analyzed in quantitative
imaging mode, a calculated excess of an isotope above its natural abundance is independent
from its location in the analyzed field. This could happen if there was some instrumental
fractionation of one isotope with respect to another, meaning that depending upon
the location over the field, the transmission for one of the isotopes is somewhat
different from another one. Control for the lack of fractionation is provided by the
image derived from isotope ratios from quantitative mass images of a set of isotopes
expected to be present at their natural abundance values and recorded over the same
field, in the same instrumental condition and in parallel, simultaneously, with the
isotope from which one expects an excess above its natural abundance at some location
within the field. This is best performed by recording together in parallel the quantitative
image of the experimental isotope and of an isotope that is expected to be present
at the natural abundance level. The beauty of the 13C/12C ratio (as in the cochlear sample labeled with 15N, Figure 3) or of the 12C15N/12C14N compared with 13C14N/12C14N (as in an adipocyte labeled with 13C, Figure 4) is that they control for the very same field that is being analyzed, and in the
same conditions. 'Normally' one is not fortunate enough to develop and use internal
controls of such high power. These controls are ideal internal controls and are immensely
more important than finding natural abundance value ratios in separate control samples
where all conditions are different from the experimental measures.

Conclusion

We have developed MIMS, combining the use of a new SIMS instrument that simultaneously
detects multiple isotopes with stable isotope labeling and quantitative image analysis.
Using MIMS, it is possible to localize and measure precisely stable or radioactive
isotopic tags in subcellular compartments in sub-cubic-micrometer volumes. Stable
isotopes can be localized simultaneously at high resolution and measured with high
precision, a unique combination. Studies with MIMS can use conventional tissue – and
cell-preparation methods. The molecular tags can be any isotopes, stable or radioactive.
One can take advantage of any molecule that has been labeled for mass spectrometry
or nuclear magnetic resonance studies (2H, 13C, 15N, 17O, 18O, 33S) or for radioactive studies (3H and 14C). These features make MIMS methodology a powerful new way of viewing and measuring,
in all fields of biomedical research, important parameters that were impossible or
difficult to see or to measure with other methods.

Quantitative image data extraction

In the scanning mode, the number of counts for each secondary ion emitted from each
pixel is stored in a computer file. Grayscale secondary-ion images for each of the
selected secondary ions are obtained after analysis by reconstructing the data onto
an array with a number of pixels equal to that of the data-acquisition array, with
the pixel intensity in the image scaled according to the number of counts detected
from that position for the ion. We developed a piece of software to extract 24-bit
quantitative image files from hundreds of raw MIMS images, keeping the counts recorded
in each pixel.

An essential feature of MIMS is the ability to locate and to quantitate regions of
the sample that have accumulated an isotope, for example 15N, after the isotope has been provided externally. This is done by deriving ratio
images. For example, the ratio image 12C15N-/12C14N- and the ratio image 13C-/12C- are derived from the pixel-by-pixel division of the respective parallel images. Quantitative
images acquired with MIMS generate far more information than can be displayed using
grayscale methods. To show high dynamic-range ratio images and to de-emphasize values
resulting from data with few counts, we developed a method based on a hue saturation
intensity transformation (HSI) [40,41] of the ratio image; HSI is derived from a calcium ratio imaging procedure [42]. Think of the pixels in a MIMS image as equivalent to test tubes. A 256 × 256 pixel
MIMS image would be an array of 65,536 test tubes from which we simultaneously count
several isotopes (for example, the abundance of 12C, 13C, 14N and 15N). The HSI method allows us to identify among the 65,536 pixel test tubes (corresponding
to areas of sub-micrometer size) those that contain significantly higher label counts.
The main advantage of HSI is to color code the value of the ratios following a scale
that can be extended or compressed, as well as taking full advantage of the fact that
humans perceive many more differences in color than differences in shades of gray.
For example, HSI enables us to identify regions of interest with significant excess
of the 15N label by a means independent of visual recognition of expected histological structures.
The use of HSI allows us to greatly simplify the first step of data analysis.

Another powerful piece of software automates the data analysis of sequences of raw
images. After creating stacks of images from analyzed fields and drawing regions of
interest, statistics for each of the regions in each of the images are automatically
tabulated and exported to the Prophet software for statistical analysis.

Isotope ratios

Regions of interest were selected using HSI ratio images; secondary-ion counts for
the same regions of interest were extracted from the corresponding unsmoothed images
of 12C-, 13C-, 12C15N- and 12C14N- and the isotope ratio values from the derived images 13C-/12C- and 12C15N-/12C14N-. The 15N/14N ratios (or 13C-/12C-) for each region of interest were also determined by dividing the total counts of
12C15N- (or 13C-) over all pixels in that region by the total corresponding counts of 12C14N- (or 12C-). Importantly, none of the image manipulations that we perform alters the counts
contained in the pixel or groups of pixels.

Nitrogen renewal

The percent nitrogen renewal was calculated for each region of interest as: 100 ×
(tissue 15N/14N ratio – control diet ratio)/(experimental diet ratio – control diet ratio), which
gives the percentage of the labeled dietary nitrogen that has been incorporated into
the sample. Zero percent represents a ratio equivalent to the terrestrial ratio; 100%
represents a ratio identical to that in the experimental diet.

Labeling and tissue preparation

All experimental protocols used in this study were approved by the Harvard Medical
Area Standing Committee on Animals, Protocol 02751. CBA/CaJ mice, 8 weeks old at the
start of the experiments, were obtained from Jackson Laboratories (Bar Harbor, ME,
USA).

Diets enriched in 15N-leucine

Standard L-leucine commercial protein-free amino acid diet (TD 86529, Harlan Teklad,
Madison, WI, USA) was used as the control diet. The experimental diet was prepared
by the manufacturer according to the same formula, but with 15N-L-leucine replacing 23% of the standard L-leucine (9 g 15N-L-leucine in the total of 38.8 g leucine, for a 3.5 kg batch of diet). To ensure
a dietary steady state on these synthetic diets, mice were placed on the control amino-acid
diet for 5 days before they were fed the experimental diet. Mice were sacrificed after
9 days or 22 days on the 15N-L-leucine diet. 15N-L-leucine (> 98% isotopically pure) was obtained from Cambridge Isotope Laboratories,
Andover, MA, USA.

The 15N/14N nitrogen isotope ratios in the diets were measured by combustion mass spectrometry
analysis (Metabolic Solutions, Nashua, NH, USA). The values were 3.657‰ (0.001 SE,
n = 12) in the control diet (equivalent to the terrestrial value) and 13.972‰ (0.573
SE, n = 12) in the experimental diet.

Mouse cochlear tissue

After 9 or 22 days on the diet, deeply anesthetized mice underwent intracardiac perfusion
with saline followed by fixative (2.5% glutaraldehyde and 1.5% paraformaldehyde in
0.065 M phosphate buffer). The temporal bones were removed, kept in fixative overnight
at 4°C, decalcified (0.1 M EDTA with 0.4% glutaraldehyde) for 3 days, rinsed in deionized
water, dehydrated in ethanol and propylene oxide, infused with propylene oxide/epon,
and embedded in epon. Other tissues were treated similarly, but not decalcified [43].

Sections from the epon-embedded tissue, 0.5 mm in thickness, were cut with glass knives
on a Sorvall ultramicrotome. The sections were deposited on (5 mm)2 silicon wafer chips, which provide a mechanically sturdy and adequately conductive
support [44]. The silicon chips were then mounted in a custom-made sample holder for insertion
into the MIMS instrument. The holder with the sections was placed in a vacuum oven
for 3 days at 50°C before analysis and then introduced in the preanalysis chamber
of the instrument in a 10-9 torr vacuum.

Teredinibacter turnerae

Cells of T. turnerae were grown in sealed 16 ml Hungate tubes containing 15.5 ml of simple salt medium
(SBM amended with 0.5% sucrose without combined nitrogen [31]) allowing a headspace of 0.5 ml of atmospheric gas. After three days the headspace
gas was removed by displacement with fresh medium and replaced with 0.5 ml of 15N gas (99%). Cells were then incubated at 22°C with gentle shaking for 0, 0.25, 0.5,
1, 2, 4, 8, 16, and 32 h. After incubation, cells were sedimented by centrifugation
(12,000 g, 10 min, 4°C) and fixed by resuspension in 500 ml 3.7% formaldehyde in seawater for
30 min. Cells were then washed by sedimentation (14,000 g, 10 min, room temperature) and resuspended first in 50% ethanol and again in distilled
water. Cells were finally resuspended in 10 μl of distilled water and 2 μl was applied
to the silica wafer and air-dried at 37°C. Cells of Enterococcus faecalis were prepared identically except that they were grown in Tryptic Soy Broth medium
(Bacto) at 37°C for 2.5 h (approximately eight doublings).

Shipworms

Shipworms, Lyrodus pedicellatus, were incubated in seawater in a 15N atmosphere, fixed in formaldehyde, embedded in epon, and sectioned for MIMS analysis.

Adipocytes

Adipocytes were prepared and analyzed in MIMS using the same methods as Kleinfeld
et al. [28].

Rat embryo fibroblasts

Rat embryo fibroblasts (REF52) were cultured in DME/F12 medium with 10% FCS. They
were serum-deprived for 26 h after they reached 70–80% confluence; pulsed for 17 h
with 10% FCS and 1 mM BrdU (1/10 of the concentration generally used in microfluorescence
studies) and 1 mM 15N-uridine; fixed in their culture dish (using formaldehyde, glutaraldehyde, and picric
acid); detached from plastic with propylene oxide; embedded in epon-Araldite; sectioned
at 0.5 μm thickness; and mounted on polished Inox rod for MIMS analysis.

For 14C analysis, REF52 cells were cultured in DME/F12 media with 10% FCS. They were serum-deprived
for 24 h after they reached 40% confluence; pulsed for 24 h with 10% FCS and 1 mCi
14C-thymidine at a concentration of 19.2 nanomol/ml; fixed in their culture dish using
formaldehyde and glutaraldehyde; detached from plastic with propylene oxide; embedded
in epon-Araldite; sectioned at 0.5 μm thickness; and mounted on silicon chips for
analysis.

Tuning for quantitative mass image acquisition

Before a MIMS experiment, the sample is first viewed and photographed under a reflection
differential interference contrast (RDIC) microscope with an automated stage, and
the coordinates of multiple regions of interest are noted and used to find the same
regions in the automated stage of the SIMS instrument. RDIC allows us to dispense
with preparing and staining alternate sections for microscopy. The sample is placed
in the analysis chamber under a vacuum of approximately 3 × 10-10 torr. A primary-ion beam of Cs+, after being shaped by the primary-ion column, is focused with the immersion lens
and strikes the sample with an energy of 16 keV (see Figure 13). The primary-ion beam scans the region of interest on the stationary sample step-by-step
in a raster pattern, generally of 256 × 256 pixels but the number of pixels can vary
in practice from 32 × 32 to 1,024 × 1,024. The secondary ions emitted from the sample
are focused with the immersion lens, shaped by the optics of the secondary-ion column,
and sent through a hexapole in the double-sector mass spectrometer made up of an electrostatic
prism followed by a magnetic prism. This permits first-order focusing of the secondary
ions as narrow lines along the focal plane of the magnet. Four detector units are
located at the focal plane and can be moved independently along it so as to select
specific atomic masses. Four electron multipliers in the detector units detect the
signals from the selected secondary ions simultaneously. The current and diameter
of the primary-ion beam, the time the beam is directed at each pixel (the dwell time),
and the size of the pixels can be adjusted according to the size of the area being
analyzed.

For each analysis, we need to ensure first that we have implanted enough Cs atoms
into the sample to maximize the secondary-ion counting statistics; second, we maximize
the secondary-ion transmission; third, we have perfect mass spectra at all masses;
and fourth, in control fields we obtain equivalent values of the isotope ratio over
the entire image field. Tuning is first done in ion-counting mode, in which the secondary-ion
count rate recorded by each detector is displayed numerically. No images are acquired.
We ensure that the responses of the electron multiplier detectors (EM; see Figure
13) are equivalent. The primary Cs+ beam is defocused and kept stationary on a standard silicon sample. The secondary-ion
current at mass 28Si is measured successively with each of the four EM detectors; the radius of the
secondary-ion beam is set to enter the appropriate EM by changing the magnetic field
strength of the magnetic prism. We adjust two parameters for each EM: the EM high
voltage is set so that the pulse height distribution (PHD, the graphical representation
of the distribution of the amplitude of the pulses generated by an EM at a particular
voltage) of the EM output is similar for all four detectors; and the EM threshold
voltage is set so that, in the absence of secondary-ion current, the noise of the
counting system is smaller than 1 count per minute. When these conditions are fulfilled,
the count rate for the same sample is the same among the four detectors, within ±
2.5%.

To begin tuning, we implant Cs atoms into the biological sample over a relatively
large field (100–150 μm) at high primary-ion current (obtained by removing the diaphragm
(D1, Figure 13) used to define the primary-beam aperture in the objective column (OC). After a period
of several minutes, Cs atoms implanted into the sample enhance the secondary-ion yield
so that the 12C14N- secondary-ion count rate reaches a steady state. Next, we ensure that the EM detectors
are located in the correct position along the focal plane of the magnet (FP, Figure
13) for each mass. We maximize the count rate of each EM by moving the detector along
the focal plane so as to maximize the secondary-ion count rate for the selected atomic
mass.

We follow with the alignment of the secondary-ion beam so that the isobars 12C15N and 13C14N, and 13C and 12C1H, are well discriminated, indicating high mass resolution, while maintaining a high
secondary-ion transmission (approximately 70–80%). An entrance slit (ES, Figure 13), whose width is a main determinant of the mass resolving power (MRP), is placed
in the path of the secondary-ion beam. We maximize the count rate for a given mass
by iteratively focusing the secondary-ion lens located in the objective column (E0S)
and by adjusting the voltages of the deflection plates P2, P3, and CY within the secondary
column. We then set an aperture slit (AS) and an energy slit (WS) to reduce angular
and energy aberrations and thus to maximize the MRP. To evaluate the MRP, we scan
the secondary-ion beam through the detectors selection slit (SS) using a set of deflection
plates (DP) and we record a small portion of the mass spectrum by plotting the secondary-ion
current versus the deflection voltage (Figure 15). The quadrupole and slit lens (LF4) are used to maximize the MRP. Then, for each
detector, using the mass spectra plots, we set the voltage of the deflection plates
(DP, Figure 13) so that the mass line of interest is centered in the selection slit (SS) of the
EM (see Figure 13). This practically eliminates any spillover from neighboring isobars.

We then switch to imaging mode, which displays 'live' scanning ion images from two
selected masses. After adjusting the size of the analyzed field, the last steps aim
to balance the tuning and to focus the primary-ion beam. We ensure that the analyzed
field is balanced, that is, that the secondary-ion intensity emitted by a small fraction
of the field is equivalent over the entire field, with iterative small voltage adjustments
of the secondary-ion objective lens (EOS), and of a series of deflection plates (P2,
P3 and CY) within the secondary column. We optimize the focus by adjusting the voltage
of the primary-ion objective lens (EOP, Figure 13). To analyze at the highest possible spatial resolution (approximately 35 nm), we
increase the demagnification of the primary-ion source by increasing the voltage of
the lens L1 (see Figure 13) in the primary column. We may then have to slightly readjust the tuning of the secondary-ion
beam. Finally, before starting analysis, we adjust the number of pixels in the image
(256 × 256 up to 2,048 × 2,048) such that the beam diameter is approximately twice
the pixel size, and we ensure that the acquisition time is long enough to obtain sufficient
counting statistics for masses with low count rates (for example 13C and 12C15N).

After a sample is introduced into the MIMS analysis chamber, we can set the instrument
to acquire qualitative MIMS images – in general at mass 12C14N, which gives a good anatomical view – within 30 minutes. Significantly more time
is required, however, for the optimization of the many instrumental parameters (tuning)
that is needed to record quantitative mass images suitable for extracting meaningful
quantitative data. For new types of samples it may take hours to a day. Yet the physics
is robust, and further tuning on an identical type of sample does not take more than
1 to 2 hours.

Additional data files

The following files are available: Additional data file 1, containing an explanation of how the volume sputtered, the useful yield, and the
detectability limit are calculated; Additional data file 2, containing an explanation of how the lateral resolution is calculated; Additional
file 3, describing data reduction from labeled and control mouse cochlear sections; Additional
data file 4, describing the relationship between the brightness of an image and the number of
counts; Additional data file 5, comparing MIMS with autoradiography; Additional data file 6, containing an explanation of the shape of mass line peaks in MIMS; and Additional
data file 7, containing more details on Figure 2e.

Additional data file 1. An explanation of how the volume sputtered, the useful yield, and the detectability
limit are calculated

Acknowledgements

Development of the new SIMS instrument was supported by ONERA, CNRS, Université Paris
Sud and Cameca (France). We thank Jean-Daniel Sraer for his help in obtaining the
prototype of the new SIMS machine and M.J. Karnovsky for his long-time support. We
thank Hjalmar Brismar and Anne Mudge for overall comments, Edmund Mroz and Tae Gee
Jung for providing mouse cochlear samples, Louise Trakimas for her most expert histological
help. Molly Palmer for her patience, efficiency and dedication in working on the manuscript,
and Anna Sharman for her comprehensive editorial attention. This work was supported
in part by NIH grants P41RR14579, P41EB001974, DC00033, DC03463, RO1DC04179, P41RR14579,
P41EB001974, R37DK39773, RO1EY12963, RO1GM47214, and RO1DK58762 and by NSF/IBN grant
IBN-998298.

References

Schoenheimer R: The Dynamic State of Body Constituents. The Edward K. Dunham Lectures for the Promotion
of the Medical Sciences. Cambridge, MA: Harvard University Press; 1942.